Project Summary Radiotherapy initiates the recruitment of alternatively stimulated (M2) macrophages to the tumor microenvironment. These cells exhibit a ?pro-tumor phenotype,? stimulating angiogenesis, suppressing anti- tumor immunity, and enhancing tumor cell radioresistence. This limits the overall efficacy of radiotherapy for solid tumors such as non-small cell lung cancer. Current research in the field of radioimmunology is focused on manipulations that either inhibit the recruitment of these cells or that alter their behavior once they reach the tumor microenvironment. However, exactly how irradiated cancer cells manipulate macrophages is largely unknown. We have discovered that extracellular vesicles elaborated by irradiated lung cancer cells (IR-EVs) induce ER-stress and elicit the production of ?pro-tumor? cytokines from macrophages in an SRA/CD204 dependent manner. The overall objective of this project is to define the molecular mechanisms by which IR-EVs induce macrophage pro-tumor cytokine expression. First, we will test the functional impact of IR-EV induced macrophage ER stress on macrophage phenotype using CHOP-/- C57BL/6 mice and soluble inhibitors of ER stress. Then, we will investigate the role of macrophage SRA/CD204 in the response of macrophages to IR-EVs. We will do this using SRA/CD204-/- C57BL/6 mice as well as a novel SRA/CD204 blocking antibody developed in our laboratory. Finally, we will compare the circulating IR-EVs of recurrent and non-recurrent non-small cell lung cancer patients to determine if they can predict the outcome of radiotherapy. EVs from recurrent and non- recurrent NSCLC patients isolated before, during, and after RT will be assessed for their ability to stimulate a pro-tumor macrophage phenotype in vitro. Then, mass spectrometry will be used to identify a potential SRA/CD204 binding protein upregulated on the surface of IR-EVs from patients and cultured cells. The potential of ligand bearing exosomes to segregate lung cancer patients into relapse or disease-free survival groups will be evaluated using a commercially available ELISA on concentrated serum exosomes.